Chromatin associated proteins

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a chromatin associated protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the chromatin associated protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the chromatin associated protein in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/092,841 filed Jul. 14, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingchromatin associated proteins in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] The human Lamin B receptor (LBR) belongs to the ERG4/ERG24 familyof nuclear envelope inner membrane proteins. It anchors the lamina andthe heterochromatin to the inner nuclear membrane. LBR can interact withchromodomain proteins. LBR has an amino-terminal domain of approximately200 amino acids followed by a carboxyl-terminal domain that is similarin sequence to yeast and plant sterol reductases. Two LBR-like geneshave recently been identified in humans which have strongcarboxyl-terminal domains of LBR and sterol reductases (Pezhman et al.(1998) Genomics 54(3):469-476). The human LBR/sterol reductase likeproteins are localized to the endoplasmic reticulum. These LBR/sterolreductase proteins may define a human gene family encoding proteins ofthe inner nuclear membrane and endoplasmic reticulum that function innuclear organization and/or sterol metabolism.

[0004] In the nucleus LBR undergoes phosphorylation by CDC2 proteinkinase in mitosis when the inner nuclear membrane breaks down intovesicles that dissociate from the lamina and the chromatin. It isphosphorylated by different protein kinases in interphase when themembrane is associated with these structures. Phosphorylation of LBRproteins may be responsible for some of the alternations in chromatinorganization and nuclear structure which occur at various times duringthe cell cycle. To date, a Lamin B receptor has not been identified inplants. A plant Lamin B receptor could be used to manipulate cell cycleregulation and plant transformability.

[0005] Accordingly, the availability of nucleic acid sequences encodingall or a portion of these proteins would facilitate studies to betterunderstand transcritional regulation, cell cycle progression, anddevelopmental events in eucaryotic cells. It would also provide genetictools for the manipulation of cell cycle regulation and increase theefficiency of transformation.

SUMMARY OF THE INVENTION

[0006] The instant invention relates to isolated nucleic acid fragmentsencoding chromatin associated proteins. Specifically, this inventionconcerns an isolated nucleic acid fragment encoding a lamin Breceptor/sterol reductase and an isolated nucleic acid fragment that issubstantially similar to an isolated nucleic acid fragment encoding alamin B receptor/sterol reductase. In addition, this invention relatesto a nucleic acid fragment that is complementary to the nucleic acidfragment encoding lamin B receptor/sterol reductase.

[0007] An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a lamin Breceptor/sterol reductase.

[0008] In another embodiment, the instant invention relates to achimeric gene encoding a lamin B receptor/sterol reductase, or to achimeric gene that comprises a nucleic acid fragment that iscomplementary to a nucleic acid fragment encoding a lamin Breceptor/sterol reductase, operably linked to suitable regulatorysequences, wherein expression of the chimeric gene results in productionof levels of the encoded protein in a transformed host cell that isaltered (i.e., increased or decreased) from the level produced in anuntransformed host cell.

[0009] In a further embodiment, the instant invention concerns atransformed host cell comprising in its genome a chimeric gene encodinga lamin B receptor/sterol reductase, operably linked to suitableregulatory sequences. Expression of the chimeric gene results inproduction of altered levels of the encoded protein in the transformedhost cell. The transformed host cell can be of eukaryotic or prokaryoticorigin, and include cells derived from higher plants and microorganisms.The invention also includes transformed plants that arise fromtransformed host cells of higher plants, and seeds derived from suchtransformed plants.

[0010] An additional embodiment of the instant invention concerns amethod of altering the level of expression of a lamin B receptor/sterolreductase in a transformed host cell comprising: a) transforming a hostcell with a chimeric gene comprising a nucleic acid fragment encoding alamin B receptor/sterol reductase; and b) growing the transformed hostcell under conditions that are suitable for expression of the chimericgene wherein expression of the chimeric gene results in production ofaltered levels of lamin B receptor/sterol reductase in the transformedhost cell.

[0011] An addition embodiment of the instant invention concerns a methodfor obtaining a nucleic acid fragment encoding all or a substantialportion of an amino acid sequence encoding a lamin B receptor/sterolreductase.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

[0012] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0013] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Chromatin Associated ProteinsSEQ ID NO: Clone Protein Designation (Nucleotide) (Amino Acid) LaminB/Sterol bms1.pk0009.e4 1 2 Reductase (corn) Lamin B/Sterolr1r2.pk0031.g9 3 4 Reductase (rice) Lamin B/Sterol wr1.pk0039.d9 5 6Reductase (wheat)

[0014] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. § 1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In the context of this disclosure, a number of terms shall beutilized. As used herein, a “nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0016] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-a-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof.

[0017] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide, are well known in theart. Thus, a codon for the amino acid alanine, a hydrophobic amino acid,may be substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0018] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize, under stringentconditions (0.1×SSC, 0.1% SDS, 65° C.), with the nucleic acid fragmentsdisclosed herein.

[0019] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent similarity of theamino acid sequences that they encode to the amino acid sequencesdisclosed herein, as determined by algorithms commonly employed by thoseskilled in this art. Preferred are those nucleic acid fragments whosenucleotide sequences encode amino acid sequences that are 80% similar tothe amino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% similar to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% similar to theamino acid sequences reported herein. Sequence alignments and percentsimilarity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0020] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0021] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0022] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0023] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0024] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0025] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic nucleotide segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a nucleic acid fragmentto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. New promoters of various types useful inplant cells are constantly being discovered; numerous examples may befound in the compilation by Okamuro and Goldberg (1989) Biochemistry ofPlants 15:1-82. It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, nucleic acid fragments of different lengths may have identicalpromoter activity.

[0026] The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

[0027] The “3′ non-coding sequences” refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0028] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0029] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0030] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0031] “Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0032] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0033] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0034] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

[0035] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0036] Nucleic acid fragments encoding at least a portion of severalchromatin associated proteins have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0037] For example, genes encoding other lamin B receptor/sterolreductase, either as cDNAs or genomic DNAs, could be isolated directlyby using all or a portion of the instant nucleic acid fragments as DNAhybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

[0038] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989)Science 243:217). Products generated by the 3′ and 5′ RACE procedurescan be combined to generate full-length cDNAs (Frohman and Martin (1989)Techniques 1:165).

[0039] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1; Maniatis).

[0040] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering chromatin organization and nuclear structurein those cells.

[0041] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

[0042] Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

[0043] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys.100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

[0044] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0045] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0046] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppresiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense synthaseenzyme genes may require the use of different chimeric genes utilizingdifferent regulatory elements known to the skilled artisan. Oncetransgenic plants are obtained by one of the methods described above, itwill be necessary to screen individual transgenics for those that mosteffectively display the desired phenotype. It is well known to thoseskilled in the art that individual transgenic plants carrying the sameconstruct may differ in expression levels; this phenomenon is commonlyreferred to as “position effect”. For example, when the construct inquestion is designed to express higher levels of the gene of interest,individual plants will vary in the amount of the protein produced andthus in enzyme activity; this in turn will effect the phenotype. Thus,in the use of these techniques their efficiency in an individualtransgenic plant is unpredictable, but given a large transgenicpopulation individuals with suppressed gene expression will be obtained.In either case, in order to save time, the person skilled in the artwill make multiple genetic constructs containing one or more differentparts of the gene to be suppressed, since the art does not teach amethod to predict which will be most effective for a particular gene.Furthermore, even the most effective constructs will give an effectivesuppression phenotype only in a fraction of the individual transgeniclines isolated. For example, WO 93/11245 and WO 94/11516 disclose thatwhen attempting to suppress the expression of fatty acid desaturasegenes in canola, actual suppression was obtained in less than 1% of thelines tested. In other species the percentage is somewhat higher, but inno case does the percentage reach 100. This should not be seen as alimitation on the present invention, but instead as practical matterthat is appreciated and anticipated by the person skilled in this art.Accordingly, the skilled artisan will develop methods for screeninglarge numbers of transformants. The nature of these screens willgenerally be chosen on practical grounds, and is not an inherent part ofthe invention. For example, one can screen by looking for changes ingene expression by using antibodies specific for the protein encoded bythe gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that the majority of samples will be negative.

[0047] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded chromatin associated protein. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 6).

[0048] All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0049] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4(1):37-41. Numerous publications describe geneticmapping of specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0050] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0051] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

[0052] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0053] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0054] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0055] cDNA libraries representing mRNAs from various corn, rice andwheat tissues were prepared. The characteristics of the libraries aredescribed below. TABLE 2 cDNA Libraries from Corn, Rice and WheatLibrary Tissue Clone bms1 Corn (Zea mays L., BMS) cell culture 1 daybms1.pk0009.e4 after subculture r1r2 Rice leaf, 15 days aftergermination, 12 r1r2.pk0031.g9 hours after infection of strainMagaporthe grisea 4360-R-62 (AVR2-YAMO) wr1 Wheat (Triticum aestivum L.)root; 7 day old wr1.pk0039.d9 seedling, light grown

[0056] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651). The resulting ESTs are analyzed using a Perkin Elmer Model377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0057] cDNA clones encoding chromatin associated proteins wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.For convenience, the P-value (probability) of observing a match of acDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Lamin BReceptor/Sterol Reductase

[0058] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs tolamin B receptor/sterol reductase from Homo sapiens (NCBI Identifier No.gi 4191396). Shown in Table 3 are the BLAST results for the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones (“FIS”):TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toLamin B Receptor/Sterol Reductase BLAST pLog Score Clone Status gi4191396 bms1.pk0009.e4 FIS 18.70 r1r2.pk0031.g9 FIS 34.52 wr1.pk0039.d9FIS 25.22

[0059] The data in Table 4 represents a calculation of the percentsimilarity of the amino acid sequences set forth in SEQ ID NOs:2, 4 and6 and the Homo sapiens sequence (SEQ ID NO:7). TABLE 4 PercentSimilarity of Amino Acid Sequences Deduced From the Nucleotide Sequencesof cDNA Clones Encoding Polypeptides Homologous to Lamin BReceptor/Sterol Reductase SEQ ID NO. Percent Similarity to gi 4191396 261% 4 46% 6 58%

[0060] Sequence alignments and percent similarity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a lamin Breceptor/sterol reductase. These sequences represent the first corn,rice and wheat sequences encoding lamin B receptor/sterol reductase.

Example 4 Expression of Chimeric Genes in Monocot Cells

[0061] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0062] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0063] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0064] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0065] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0066] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0067] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

[0068] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0069] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0070] Soybean embroys may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0071] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0072] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0073] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0074] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0075] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0076] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

[0077] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0078] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0079] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25° C. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

1 7 1 413 DNA Zea mays 1 gcacgagcgg agacctgctg ctagcacttt cgttcagcttgccctgtgga gtgagttccg 60 tggtcccata cttctacccc acgtacctgc tcattctactggtcttgagg gaaaggcgcg 120 atgaggcgag gtgctcgcag aagtacaggg agatctgggcagagtactgc aagctcgtgc 180 cgtggaggat cctgccttat gtgtactgaa gagacggtagaaaccaaggc agctcatggc 240 cctgggccag ctgtaaacct tattttgttt gcccttaaccagttggtgaa tgttgatgta 300 gcactcggta aactgtgacc gtgcaaactt ttgttattgttggtccatac atgtttggaa 360 tcgtgaatca gaccgcctca cttggtggca aaaaaaaaaaaaaaaaaaaa aaa 413 2 68 PRT Zea mays 2 Thr Ser Gly Asp Leu Leu Leu AlaLeu Ser Phe Ser Leu Pro Cys Gly 1 5 10 15 Val Ser Ser Val Val Pro TyrPhe Tyr Pro Thr Tyr Leu Leu Ile Leu 20 25 30 Leu Val Leu Arg Glu Arg ArgAsp Glu Ala Arg Cys Ser Gln Lys Tyr 35 40 45 Arg Glu Ile Trp Ala Glu TyrCys Lys Leu Val Pro Trp Arg Ile Leu 50 55 60 Pro Tyr Val Tyr 65 3 604DNA Oryza sativa 3 gcacgagatc actgggatgg tggcttttga gaaacaaagtggagctgtcc cttttggctg 60 ctgtagttaa ctgcttcatt ttcgttattg gctatcttgtgttcagagga gccaacaaac 120 aaaaacatat cttcaagaag aaccctaaag ctcttatttggggtaaacct cccaaacttg 180 tcggggggaa gctacttgta tctggctact ggggaattgcaaagcactgc aattatcttg 240 gggatatact gctagctctt tcatttagct taccctgtggaaccagttcg gtgatcccat 300 acttctaccc aacatacctg ttcattttgc tgatatggagggaacgaagg gacgaagcaa 360 ggtgctcaga gaagtacaag gagatctggg tagaatattgcaagcttgtg ccttggagga 420 tctttcctta cgtgtattaa atccaaatat tttgcctagcaggtgcatcg ttgtagaacc 480 aagagcgttg ttgtgctatt tgaacatgta aaattcaccaagattcctgt tgtttatttg 540 tagctgacat ccgtgttgaa tatcaattaa catagattttgttgaaaaaa aaaaaaaaaa 600 aaaa 604 4 145 PRT Oryza sativa 4 Thr Arg SerLeu Gly Trp Trp Leu Leu Arg Asn Lys Val Glu Leu Ser 1 5 10 15 Leu LeuAla Ala Val Val Asn Cys Phe Ile Phe Val Ile Gly Tyr Leu 20 25 30 Val PheArg Gly Ala Asn Lys Gln Lys His Ile Phe Lys Lys Asn Pro 35 40 45 Lys AlaLeu Ile Trp Gly Lys Pro Pro Lys Leu Val Gly Gly Lys Leu 50 55 60 Leu ValSer Gly Tyr Trp Gly Ile Ala Lys His Cys Asn Tyr Leu Gly 65 70 75 80 AspIle Leu Leu Ala Leu Ser Phe Ser Leu Pro Cys Gly Thr Ser Ser 85 90 95 ValIle Pro Tyr Phe Tyr Pro Thr Tyr Leu Phe Ile Leu Leu Ile Trp 100 105 110Arg Glu Arg Arg Asp Glu Ala Arg Cys Ser Glu Lys Tyr Lys Glu Ile 115 120125 Trp Val Glu Tyr Cys Lys Leu Val Pro Trp Arg Ile Phe Pro Tyr Val 130135 140 Tyr 145 5 572 DNA Triticum aestivum 5 tacttgtatc tggctactggggcattgcaa ggcactgcaa ttaccttgga gatctgcttc 60 tggcactctc attcagcttgccttgtggag ccagctccgt gatcccgtac ttctacccga 120 cctacctgct gatcctgctgatatggagag aacgaagaga cgaggcgagg tgctcagaga 180 agtacaagga catctgggcagagtactgca agcttgtgcc ctggaggatt ctaccttacg 240 tgtactgatt agttaaagaaccagaaggcc atgttgtatt gttgtttttg gccctgatga 300 tcctgcataa ctaaatggtaaggtcttttg tacgtttttc ttggatatcc agttttaaat 360 tgaagctgca tcgatcttttagctttgttg gggaagtgct gctaattttc atttgagctg 420 tccctttttt tcttcatccccttctattgc tgaaagaaga gaataccgtt gggaaaaaaa 480 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaat 540 aaaaaaaaat ctcgaggggggcgccgtacc ca 572 6 81 PRT Triticum aestivum 6 Leu Val Ser Gly Tyr TrpGly Ile Ala Arg His Cys Asn Tyr Leu Gly 1 5 10 15 Asp Leu Leu Leu AlaLeu Ser Phe Ser Leu Pro Cys Gly Ala Ser Ser 20 25 30 Val Ile Pro Tyr PheTyr Pro Thr Tyr Leu Leu Ile Leu Leu Ile Trp 35 40 45 Arg Glu Arg Arg AspGlu Ala Arg Cys Ser Glu Lys Tyr Lys Asp Ile 50 55 60 Trp Ala Glu Tyr CysLys Leu Val Pro Trp Arg Ile Leu Pro Tyr Val 65 70 75 80 Tyr 7 418 PRTHomo sapiens 7 Met Ala Pro Thr Gln Gly Pro Arg Ala Pro Leu Glu Phe GlyGly Pro 1 5 10 15 Leu Gly Ala Ala Ala Leu Leu Leu Leu Leu Pro Ala ThrMet Phe His 20 25 30 Leu Leu Leu Ala Ala Arg Ser Gly Pro Ala Arg Leu LeuGly Pro Pro 35 40 45 Ala Ser Leu Pro Gly Leu Glu Val Leu Trp Ser Pro ArgAla Leu Leu 50 55 60 Leu Trp Leu Ala Trp Leu Gly Leu Gln Ala Ala Leu TyrLeu Leu Pro 65 70 75 80 Ala Arg Lys Val Ala Glu Gly Gln Glu Leu Lys AspLys Ser Arg Leu 85 90 95 Arg Tyr Pro Ile Asn Gly Phe Gln Ala Leu Val LeuThr Ala Leu Leu 100 105 110 Val Gly Leu Gly Met Ser Ala Gly Leu Pro LeuGly Ala Leu Pro Glu 115 120 125 Met Leu Leu Pro Leu Ala Phe Val Ala ThrLeu Thr Ala Phe Ile Phe 130 135 140 Ser Leu Phe Leu Tyr Met Lys Ala GlnVal Ala Pro Val Ser Ala Leu 145 150 155 160 Ala Pro Gly Gly Asn Ser GlyAsn Pro Ile Tyr Asp Phe Phe Leu Gly 165 170 175 Arg Glu Leu Asn Pro ArgIle Cys Phe Phe Asp Phe Lys Tyr Phe Cys 180 185 190 Glu Leu Arg Pro GlyLeu Ile Gly Trp Val Leu Ile Asn Leu Ala Leu 195 200 205 Leu Met Lys GluAla Glu Leu Arg Gly Ser Pro Ser Leu Ala Met Trp 210 215 220 Leu Val AsnGly Phe Gln Leu Leu Tyr Val Gly Asp Ala Leu Trp His 225 230 235 240 GluGlu Ala Val Leu Thr Thr Met Asp Ile Thr His Asp Gly Phe Gly 245 250 255Phe Met Leu Ala Phe Gly Asp Met Ala Trp Val Pro Phe Thr Tyr Ser 260 265270 Leu Gln Ala Gln Phe Leu Leu His His Pro Gln Pro Leu Gly Leu Pro 275280 285 Met Ala Ser Val Ile Cys Leu Ile Asn Ala Ile Gly Tyr Tyr Ile Phe290 295 300 Arg Gly Ala Asn Ser Gln Lys Asn Thr Phe Arg Lys Asn Pro SerAsp 305 310 315 320 Pro Arg Val Ala Gly Leu Glu Thr Ile Ser Thr Ala ThrGly Arg Lys 325 330 335 Leu Leu Val Ser Gly Trp Trp Gly Met Val Arg HisPro Asn Tyr Leu 340 345 350 Gly Asp Leu Ile Met Ala Leu Ala Trp Ser LeuPro Cys Gly Val Ser 355 360 365 His Leu Leu Pro Tyr Phe Tyr Leu Leu TyrPhe Thr Ala Leu Leu Val 370 375 380 His Arg Glu Ala Arg Asp Glu Arg GlnCys Leu Gln Lys Tyr Gly Leu 385 390 395 400 Ala Trp Gln Glu Tyr Cys ArgArg Val Pro Tyr Arg Ile Met Pro Tyr 405 410 415 Ile Tyr

What is claimed is:
 1. An isolated nucleic acid fragment encoding alamin B receptor/sterol reductase comprising a member selected from thegroup consisting of: (a) an isolated nucleic acid fragment encoding anamino acid sequence that is at least 65% similar to the amino acidsequence set forth in a member selected from the group consisting of SEQID NO:2, 4 and 6; (b) an isolated nucleic acid fragment that iscomplementary to (a).
 2. The isolated nucleic acid fragment of claim 1wherein nucleic acid fragment is a functional RNA.
 3. The isolatednucleic acid fragment of claim 1 wherein the nucleotide sequence of thefragment comprises the sequence set forth in a member selected from thegroup consisting of SEQ ID NO:1, 3 and
 5. 4. A chimeric gene comprisingthe nucleic acid fragment of claim 1 operably linked to suitableregulatory sequences.
 5. A transformed host cell comprising the chimericgene of claim 4 .
 6. A lamin B receptor/sterol reductase polypeptidecomprising all or a substantial portion of the amino acid sequence setforth in a member selected from the group consisting of SEQ ID NO:2, 4and
 6. 7. A method of altering the level of expression of a chromatinassociated protein in a host cell comprising: (a) transforming a hostcell with the chimeric gene of claim 4 ; and (b) growing the transformedhost cell produced in step (a) under conditions that are suitable forexpression of the chimeric gene wherein expression of the chimeric generesults in production of altered levels of a chromatin associatedprotein in the transformed host cell.
 8. A method of obtaining a nucleicacid fragment encoding all or a substantial portion of the amino acidsequence encoding a chromatin associated protein comprising: (a) probinga cDNA or genomic library with the nucleic acid fragment of claim 1 ;(b) identifying a DNA clone that hybridizes with the nucleic acidfragment of claim 1 ; (c) isolating the DNA clone identified in step(b); and (d) sequencing the cDNA or genomic fragment that comprises theclone isolated in step (c) wherein the sequenced nucleic acid fragmentencodes all or a substantial portion of the amino acid sequence encodinga chromatin associated protein.
 9. A method of obtaining a nucleic acidfragment encoding a substantial portion of an amino acid sequenceencoding a chromatin associated protein comprising: (a) synthesizing anoligonucleotide primer corresponding to a portion of the sequence setforth in any of SEQ ID NOs:1, 3 and 5; and (b) amplifying a cDNA insertpresent in a cloning vector using the oligonucleotide primer of step (a)and a primer representing sequences of the cloning vector wherein theamplified nucleic acid fragment encodes a substantial portion of anamino acid sequence encoding a chromatin associated protein.
 10. Theproduct of the method of claim 8 .
 11. The product of the method ofclaim 9 .